Rotaviruses (RVs) are the most common cause of severe gastroenteritis in young children worldwide. We propose to continue our investigations of important unanswered questions concerning: the basis of RV host range restriction, the regulation of innate immunity, and the targets of acquired heterotypic protection. We will carry out our studies using biologically relevant whole animal and primary human intestinal epithelial organoid systems with a focus on the factors that can be demonstrated to act in vivo or ex vivo. We now have several powerful analytic tools to facilitate these investigations including single cell transcriptional assays, mass cytometry (CyTOF), a well characterized murine model of homologous and heterologous rotavirus infection and an ex vivo RV replication assays in human organoids. Specifically, we propose to: 1) Identify the mechanisms underlying host range restriction of RVs. RV replication in vitro is regulated by the RV NSP1 protein, which modulates several components of the IFN signaling cascade in vitro. The relevance of these in vitro effects to RV replication in vivo or ex vivo has not been carefully examined. We will use single-cell transcriptional and novel protein analytical tools to evaluate viral replication in differentiated human intestinal epithelial cells (IECs) and suckling mice infected with RVs expressing various homologous and heterologous RV proteins to identify host factors involved in protecting IECs from heterologous RV attack. 2) Determine the cellular origin of IFN responses to RV in the intestine and characterize the effects of these responses on RV local and systemic replication in suckling mice and human intestinal organoids. Previous findings suggest that types I, II, and III IFNs play only limited roles in restricting homologous murine RV replication in mice but are critical to the restriction of heterologous (non-murine) RV strains in the gut. We will extend thes findings and determine the cellular origin of the various IFN responses in vivo, the mechanism by which they are induced, and the relevance of the individual IFNs to homologous and heterologous RV replication in young mice in intestinal epithelial cells and at systemic sites. 3) Identify the targets of heterotypic humoral immunity to RV in people. Heterotypic (serotype cross- reactive) immunity is critical for preventing recurrent RV disease. We will identify, at the immunoglobulin (Ig) molecule level, the viral protein targets of heterotypic immunity in people. Our hypothesis is that humans circumvent the serotypic diversity of naturally circulating RVs by expressing Ig molecules directed at RV VP4 and/or VP7 epitopes that mediate heterotypic as well as homotypic protection. We also hypothesize that non-neutralizing IgA antibodies to RV VP6 can mediate heterotypic intracellular neutralization during transcytosis through IECs based on prior mouse and in vitro studies. We will now use an intestinal organoid system to directly determine whether this mechanism is operative in the human small intestine.